Fractionation of protein mixtures

ABSTRACT

This invention relates to the separation of complex protein mixtures by lowering (desalting) or by raising (salting-out) their electrolytic (ionic) concentration by electrodialysis followed by chilling, pH adjustment, filtration, and/or centrifugation and optionally thereafter restoration of the lost electrolyte or removal of the gained electrolyte and water. This technique is especially adaptable for therapeutic plasma exchange performed in situ for immunepheresis i.e. removal of globulin or immunecomplexes implicated in autoimmune diseases. The techniques are also applicable for antihemophilic factor separation and purification, and separation of proteins from whey.

This application is a continuation-in-part of parent application Ser.No. 111,144 filed Jan. 10, 1980 now U.S. Pat. No. 4,276,140.

BACKGROUND OF THE INVENTION

Biological fluids such as blood plasma or serum, milk whey, urine, etc.contain a mixture of several proteins. For example, blood plasmacontains albumin (3.5-4.5 g/100 ml, M. wt 66,000), fibrinogen (0.20-0.45g/100 ml, M. wt 340,000), α-globulins (0.4-1.0 g/100 ml) β-globulins(0.8-1.8 g/100 ml, M. wt. 160,000), IgM (0.06-0.25 g/100 ml, M. wt.950,000), etc. (Frank W. Putnam, The Trace Components of Plasma, AnOverview). The immunoglobulins (Ig's) are very important since they areinvolved in the protective and defensive mechanisms against infectiousorganisms. Clinical diseases characterized by imbalances of thesesystems of proteins for example either in the ability to recognizeinvading organisms or to recognize indigenous protein or polynucleicacids, have promoted the basic understanding of the clinical aspects ofthe science of immunity. Abnormal immunological reactions are now knownto cause a wide spectrum of diseases. Examples of diseases known to beassociated with immune complex reactions include, for example, serumsickness, glomerulonephritis and myasthenia gravis. Plasmapheresis is atechnique used to curtail, favorably interfere with or stop theimmunopathologic process associated with circulating humoral antibodyand/or immune complexes of the plasma. [Glassman, Rationale forPlasmapheresis, "Plasma Therapy" Vol. 1 No. 1, Page 13 (1979)].

A known method is to plasmapherese about 4 liters of blood bycentrifugation or cross-flow filtration over a period of 2-4 hours. Theplasma removed from the patient in this way is usually discarded andreplaced by albumin and either physiological saline or Ringer's solutionto make up the protein, electrolyte, and water balance. This is anexpensive method. In another method the replacement of the removedplasma is accomplished by giving fresh or frozen pool plasma, and thoughless expensive, suffers from the risk of transmitting hepatitis virus tothe patient. The method of the present invention (referred to asimmunepheresis) overcomes these problems by selectively removingimmuneglobulins, euglobulins or euglobulin antigen complexes causing orresulting from the disease and at the same time restoring the majorportions of albumin, electrolyte (salt) and water and thus returning tothe patient his or her own plasma (substantially depleted in Ig or Igantigen complex) containing the proper protein, risk free from hepatitissince no additional albumin or donor plasma is required.

Antihemophilic factor (AHF) or antihemophilic globulin (Factor VIII, AHFor AHG) is one of the constituents involved in the coagulation of blood.A hereditary disorder of blood coagulation, hemophilia, results inprofuse bleeding in joints, muscles or internal organs as a result orminor trauma. This disease appears to be due to a deficiency of aspecific plasma protein AHF. Affected individuals frequently requiretherapy following minor accidents. In case surgery is required, clottingabnormality is corrected by fresh plasma transfusions or by injection ofFactor VIII concentrate, the latter being preferred since it avoidshyperproteinemia and possible kidney dysfunction resulting from largevolume transfusions.

Prior art methods for production of AHF consist for example, of takingpool-plasma, forming a cryoprecipitate, centrifuging the precipitatewhich mainly consists of a mixture of AHF and fibrinogen, removingfibrinogen and thereafter employing lyophilization to produce AHFconcentrate. These methods suffer from the disadvantages of being longand cumbersome and of having the risk of transmitting hepatitis becauseof the pool-plasma source. Also the presence of fibrinogen as animpurity makes it difficult for the AHF concentrates to go intosolution. In addition, due to an elapse of several days between donationand use there is a considerable loss of AHF activity. An AHF unit isdefined as the activity present in 1 ml. of average normal pooled humanplasma which is less than 1 hour old (100% AHF level). Thus after sixhours the loss in activity in extra corporeal liquid plasma can be asgreat as 80%. A rapid method of processing AHF would prevent this lossof activity. The apparatus and methods of the present invention overcomethese problems by being suited to an on-line real-time method. Thereforethe recovery of AHF can be as high as 4 to 5 times that of the present,long elapsed time methods. The present invention is adaptable to asmaller pooled source, e.g. 2-3 hepatitis-free members of thehemophiliac's family can donate plasma and have the AHF recovered onsite within a short time thereafter thus providing a hepatitis-free AHFof very high activity. On-line methods of this invention can also beused to recover Factor VIII from donors during plasmapheresis.

The basic techniques employed in the present invention, i.e.plasmapheresis and electrodialysis are each well known in the prior art.The novel combination of the techniques described herein produces asynergism i.e. it increases the efficacy of each step and of thecombination in an unexpected manner and makes them extremely usefulespecially for in situ real-time therapeutic use for patients for whomremoval of Ig's or complexes thereof is required.

The methods of the present invention will be described using plasma andwhey proteins as preferred examples but the scope of this invention canalso be applied to other biological fluids or other proteins withoutlimiting the scope of the invention. The use of electrodialysis forsalting-out or alternatively desalting to obtain protein separations canserve as very efficient tools in the hands of protein chemists.

THE INVENTION

The present invention relates to the application of electrodialysis forseparating aqueous protein mixtures into fractions having intrinsicallydistinguishable compositions as determined by well known physical orchemical procedures. The invention involves the fractionation or partialresolution of protein mixtures and restoration thereafter of their saltand water balances. It relates not only to the fractionation by saltdepletion (desalting) but also by salt addition (salting-out). Theprotein mixtures comprise principally (but not exclusively) plasma,serum or their derivative fractions. The electrodialysis processemployed in desalting removes dissolved salts (ions) and consequentlyeuglobulins or their complexes are substantially precipitated by thereduction of ionic concentration, if desired combined with temperatureand/or pH changes. Upon removal of salts, the interaction of the saltions with the ionizable groups of the proteins is apparently reduced,allowing interaction among the euglobulin molecules hence precipitatingthem. Albumin and other proteins which are not euglobulins in nature donot precipitate at the (low) salt concentrations which are effective foreuglobulins and therefore remain in solution for subsequent return tothe patient or for recovery. After removal of euglobulin turbidity (orprecipitate) the ionic concentration of the plasma may optionally bereturned to substantially its initial value by using the salt depletedplasma as the salt receiving stream in an electrodialysis stack ormodule. The salt depleted plasma is thus substantially restored inelectrolytes (and water) and can be given back to the donor or to thepatient without any further modification of the salt or water content.

In the salting-out embodiment of the process, salt is brought into theprotein mixture to cause the various proteins to precipitate outone-by-one as the ionic strength increases. The salting-out agents inthis group apparently operate by decreasing the activity of the water inthe solvent mixture, thereby dehydrating the hydrophilic groups of theprotein molecules and thereby causing precipitation of proteins.

In a third embodiment of this invention, use is made of the addition ofcertain agents e.g. metal ions, small anions and polyanions(polyphosphates etc.) which tend to cause precipitation (turbidity)apparently by a different mechanism whereby the electrostatic charges ofa few critical groups on the protein seem to be effected. Sinceionization of these critical groups is required to maintain a normalstate of hydration of the protein molecule, precipitation is ofteninduced by the mere effect of compensating the net electrical charge ofthe of the protein molecules. Such agents are needed only in low anddefinite concentrations since the mechanism is not a bulk effect.Electrodialysis can accomplish this in an excellently controlled manner.

The prior art utilized direct bulk addition of these agents thus causingpowerful localized effects. With electrodialysis, all the abovedescribed embodiments of the present invention can be handled easily andfractionation can be easily controlled or in some cases even enhanced.

More specifically, certain embodiments of the present invention compriseprocesses for fractionating liquid protein mixtures containing dissolvedsalt or/salts therein by employing electrodialysis (ED) apparatus havingone or more pairs of salt receiving and salt diluting chambers,separated from each other by ion-selective, neutral (non-selective) orcombination of neutral and ion-selective membranes. In one embodiment,electric current is impressed between end electrodes to reduce the saltcontent of a protein mixture located in the salt diluting chambers bytransfering the salts from such chambers to adjacent receiving chambers.Such desalting is continued until turbidity is produced. The productionof turbidity may be facilitated if desired by prior, simultaneous orsubsequent alteration of pH and/or temperature. Substantially desaltedprotein mixture from the diluting chambers is collected and treated toseparate and remove therefrom one or more of the protein componentscausing turbidity. Optionally thereafter the resulting salt depletedprotein mixture is optionally passed into the salt receiving chamberswhereby the salts entering such chambers from the adjacent dilutingchambers will substantially restore to the desalted protein mixture itsoriginal salt and water content. Such renormalization is desirable ifthe if the protein mixture is blood plasma which it is desired to returnto the donor.

The process described above its especially efficacious where the liquidprotein mixture is blood plasma or serum, where the protein componentsremoved are globulins and/or their complexes and in which at least oneof the membranes in every pair is ion-selective.

An alternative method of practicing the above described embodimentwherein at least one of the membranes in every pair is ion selective isto collect the desalted protein mixture from the diluting chambers ofthe ED stack, remove one or more of the precipitated proteins from thedesalted protein mixture and thereafter recycle the resulting saltdepleted, protein depleted mixture back into the prior dilutingchambers. A direct current of such polarity is applied so that the priordiluting chambers now containing a salt depleted mixture become saltconcentrating or receiving chambers and the former receiving chamberscontaining salts removed in the first part of the procedure becomediluting or salt depleting thus substantially restore the original saltand water content of the desalted protein mixture.

In another embodiment of the invention, the said precipitation orturbidity may be caused by salting-out i.e. by the addition to theprotein mixture of a salting-out agent. These agents include forexample, sulfate salts (Na₂ SO₄, K₂ SO₄, (NH₄)₂ SO₄, MgSO₄ etc.),acetate salts (sodium or potassium acetate etc.) citrate salts (sodiumor potassium citrate etc.) chloride salts (NaCl, KCl, MgCl₂, CaCl₂,LaCL₃ and other substantially non-toxic, soluble salts. Theelectrodialysis apparatus used is similar to the first described case;the salt enriched protein mixture in which turbidity has occurred due tosalting out by an agent, for example, Na₂ SO₄, is collected and treatedto separate and remove therefrom the one or more of the proteincomponents which cause turbidity. The salt supplying (depleting) streamwill contain salting-out agent or alternatively the salt enriched plasmaobtained after removal of the precipitate. Thus at essentially the sametime, both operations can be accomplished i.e. removal of theprecipitating or salting-out agent and the addition of the said agent tocause salting-out. The salt depleted protein stream may then be sentback to the source, optionally after making up the electrolyte balanceto substantially its original salt and water content.

The salting-out process described above is especially adaptable wherethe liquid protein mixture is blood plasma or serum and where theprotein components removed are γ-globulins and/or their complexes. EDcan add the precipitating agent at a controlled rate, a very importantfactor. A slow rate of addition of the precipitating agent leads toformation of crystalline protein precipitates of greater protein puritywhich have far less absorbed contaminants compared with the finer flocksof proteins carried down by the rapid addition of the precipitant whichmay contain proteins which would not precipitate upon slow addition.

In still another embodiment of this invention the precipitation may becaused by alteration of pH by ED to bring the pH to substantially theiso-electric point (pI) of a certain group of proteins e.g. γ-globulinsin case of blood plasma. Salting-out can be accomplished at a lower saltconcentration if operated near the isoelectric point of the protein.Precipitation by desalting can be accomplished at a higher saltconcentration if operated near the isoelectric point.

In a further embodiment of this invention, protein fractionation canalso be carried out by the addition of e.g. zinc glycinate (finalconcentration of about 20 mM) at a pH of about 7.2 by ED. The zinc ion(Zn⁺⁺) causes precipitation of the various proteins (γ-globulins,fibrinogen, etc.) in plasma without causing removal of albumin. Thismethod is similar to the above mentioned salting-out embodiment in thatthe diluting compartment is freed of zinc salt and the concentratingcompartment containing the protein mixture is enriched to causeprecipitation. The amount of Zn⁺⁺ needed is very small when compared tomany other salting-out agents since the mechanism of precipitationapparently consists in merely compensating the net negative electricalcharge of the molecule; the balance of charges of the remainingionogenic groups being zero, the essentially neutral protein molecule isapparently not capable of attracting sufficient amounts of water toremain in solution.

Some of the techniques and embodiments described hereinabove may becombined which will be obvious to those skilled in the art. For examplein the separation of certain γ-globulins from plasma, a direct additionof salting-out agent can be considered in combination withelectrodialysis to remove the added salt to recover a relatively richalbumin solution (after removal of globulin precipitate).

In the examples Na₂ SO₄ is employed as the preferred salting-outprecipitating agent but this should not be considered as limiting. Othersalts can be used and also their mixtures to refine the fractionationprocess.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a schematic view of an on line apparatus for treating aprotein solution for the selective removal of proteins byelectrodialysis desalting and then rebalancing the salt contentsimultaneously in the same electrodialysis stack.

FIG. 2 is a schematic view similar to FIG. 1 but where the rebalancingof the salt content is accomplished using polarity reversal.

FIG. 3 is a schematic view of an on line apparatus for treating aprotein solution for selective removal of proteins by salt additionusing electrodialysis and then removal of the excess salt simultaneouslyin the same electrodialysis stack.

FIG. 4 is a schematic view illustrating a system where the salt is addeddirectly to cause selective precipitation of proteins followed byremoval of the excess salt by electrodialysis.

FIG. 5 is a graph which compares the results of adding Na₂ SO₄ byvarious methods and also shows the results obtained by the addition of asalt mixture on the selective removal of two protein components; and

FIG. 6 is a schematic sectional view of an electrodialysis stack usingAC current and neutral membranes in combination with ion exchangemembranes.

DETAILED DESCRIPTION

Electrodialysis (ED) is widely practiced for desalting of aqueoussolutions: brackish water, whey milk (U.S. Pat. Nos. 3,433,726;3,447,930; 3,595,766; 3,757,005; 3,754,650 etc.). These patents areconcerned only with reducing the salt content of a liquid rather thanusing the ED process in a complex scheme of fractionating andsubsequently rebalancing the salt and water content of a mixture ofproteins intended for example, for therapeutic use as in cases ofplasmapheresis.

Desalting by ion exchange column technology has been used in the past tocause precipitation and thus fractionation of plasma proteins (U.S. Pat.Nos. 3,234,199; 3,073,744). This process however has limited flexibilityand the columns are difficult to handle, clean and sterilize whenemployed under conditions necessary for protein fractionation.

It has now been discovered that electrodialysis can be used not only inthe fractionation of proteins as a result of desalting, but also can beemployed in a salting-out process and also in a process to restore theelectrolyte (salt) and water balance of the resulting processed proteinmixtures. The resulting protein is thus ready to be returned to thedonor or to a patient with substantially its original salts. Thecombination of the techniques outlined herein include as essential stepsthe electrodialysis of the protein mixture, (optionally combined withtemperature and pH alteration) and separation of certain precipitatedproteins, thereafter the optional substantial restoration of the saltand water balance of the original mixture. This novel method increasesthe efficacy of each step in an unexpected manner and makes the processextremely useful especially for in situ real-time therapeutic use forplasmapheresis patients where removal of globulins or their complexes isrequired along with the restoration of essentially the original plasma.By this method, not only is the expense of albumin and salt replacementavoided but also the risk of transmitting hepatitis inherent in thegiving of fresh or frozen pool plasma.

In a first embodiment of fractionation by desalting, process andapparatus will be hereinafter described by referring to FIGS. 1 and 2where like parts are referenced with like numerals. In the Figures thefluid under treatment is described with respect to blood plasma but itcan be understood to be any other protein mixture. As shown in FIG. 1,citrated or heparinized blood (1) is ultrafiltered and/or centrifuged(2) to separate out the cell components (3), or any other suspension[referred to as formed elements (FE)] and the remaining plasma (15) issent to an electrodialysis (ED) stack (4) such as that commerciallyavailable from Ionics, Inc. Watertown, MA. Electrodialysis equipment andtheir methods of operation are more fully descirbed in U.S. Pat. Nos.2,848,403; 2,863,813; 3,003,940; 3,341,441; 4,115,225 and others. Such astack normally comprises one or more pairs of concentrating and dilutingchambers separated by alternating anion and cation exchange membranes.Ion selective membranes can also be replaced under some circumstances byessentially electrically neutral membranes. Thus the anion membrane canbe replaced by a neutral membrane if reduced current efficiency forionic transfer can be tolerated. The chambers are located between anode(+) and a cathode (-). An electrolyte solution is preferably passedthrough the cathode and anode chambers to conduct current across theconcentrating and the diluting chambers. The electric current is passeduntil at least incipient turbidity is produced, or until such turbiditywill be produced when the temperature is reduced and/or the pH isadjusted to substantially the pI of the least soluble protein. Usually aconcentrating chamber isolates the electrode solutions from the productor diluting chambers. The membranes are generally but not necessarilyselected so as to minimize transfer of low molecular weight compoundssuch as blood sugars. The flow rates through the stack and the appliedelectric current are regulated so that excessive changes in pH areavoided. Plasma is passed into and through the diluting chambers and byimpressing a direct current across the electrodes, the salt or ioniccontent of the plasma is reduced due to the passage of salt into theadjacent concentrating chambers (note vertical arrows in stack) whichchambers may be primed if desired initially with a small amount ofplasma or albumin. The resulting substantially desalted plasma (5) iscollected from the diluting chambers (not shown) and passed into meansfor separating and removing one or more proteins forming turbidity(globulins or their complexes in this case). The separating means may,for example, consist of a heat exchanger (6) to lower temperatures, pHadjustment and centrifuging and/or ultrafiltration apparatus (7). Afterremoval of the turbidity or precipitated globulins (17) or otherproteins, the salt depleted mixture (8) is passed into and through theconcentrating chambers (not shown) of the ED stack (4) thereby allowingit to receive the salts from the adjacent diluting chambers (notevertical arrows) and hence restoring substantially the original saltcontent of the mixture. This salt restored mixture (9) is nextoptionally passed through a heat exchanger (10) to adjust the mixture toapproximately body temperature where necessary, and then supplied withthe formed elements (e.g. red and white cells and platelets) (3)previously separated from the plasma. This restored protein mixture (12)can then be given back to the patient (14) substantially without outsideaddition of albumin or electrolyte. Thus this process is essentiallyclosed, self sufficient, and capable of in-situ real-time operation fortherapeutic plasma exchange.

If the temperature of the plasma during electrodialysis is maintained inthe range of about 0° to 40° C., if the velocity of the protein mixturein the diluting chambers is in the range of 3 to 40 cm/sec. and theratio of current density (CD) in ma/cm² to protein solution conductivity(K) in milli Siemens/cm,(CD/K) is kept in the range of 0.1-10, pHchanges in the protein will not be substantial and the precipitate soformed (even on relatively complete desalting) will be such as tosubstantially avoid plugging the chambers of the ED stack. (It should benoted that one mille Siemen equals one milli mho.

Another embodiment of the apparatus and process of this invention isshown in FIG. 2. The fluid is again plasma (1) but can be any otheraqueous, fluid protein mixture. (citrate or heparin may or may not beadded to minimize plasma coagulation during processing.) The plasmawhether or not heparinized or citrated is ultrafiltered or centrifuged(2) to remove turbidity and then sent to ED stack (4) similar to thatdescribed in Example 1. Plasma is introduced into the diluting chambers(not shown) and on passing a direct current across the stack, salts fromthe plasma are transferred (note vertical arrows) to the concentratingchambers. The salt depleted mixture (5) from the diluting chambers ispassed through heat exchanger (6) to chill the plasma and theprecipitate formed (17) is separated by an ultrafilter or a centrifuge(7) or similar device. The desalted supernatant (8) is then fed to thesalt concentrating chambers (not shown) of an electrodialysis stack(18). For this second ED operation another ED stack (18) is shown, inpractice it can be the original ED stack (4) where the formerconcentrating stream (19) of the ED stack (4) forms the diluting stream.The polarity for ED stack (18) may be the reverse of ED stack (4). Thissecond stage ED (18) causes the salts from the former concentratingstream (19) to return to the desalted plasma whereby the salt balance isrestored. This renormalized plasma (9) is then passed through heatexchanger (10) and the blood cells or FE (3) are then added. The thusprocessed blood can be given back to the donor or other patient (14).

EXAMPLE I

This example illustrates the restoration of the electrolyte and waterbalance of a desalted plasma using a fresh plasma in the dilute stream.

Apparatus used was a laboratory electrodialysis stack using only onecell pair (i.e. one diluting and one concentrating chamber defined byion-selective membranes) located between terminal electrode chambers. A0.2 N Na₂ SO₄ solution was used for the electrode streams to conduct thedirect current. A volume of 360 ml of citrated otherwise fresh plasmawas used in the diluting stream and 340 ml of desalted plasma was usedin the concentrating stream. The linear velocity of the diluting streamwas about 25 cm/sec., the temperature was maintained at 15° to 20° C.and the flow rates at 90 ml.min per cell pair. The effective cell areawas about 220 cm². The progress of the run is summarized in thefollowing table:

    ______________________________________                                                    Diluting stream                                                                           Conc. stream                                                      (citrated fresh                                                                           (desalted plasma)                                                 plasma)     Con-                                                                        Conduc-         duc-                                                          tivity          tivity                                        Time            values          values                                  CD/K  (min.)  Amps    (K)    pH  vol. (K)   pH  vol.                          ______________________________________                                        4.7   0       17      16.500 8.2 360   0.030                                                                              5.2 340                           4.7   6       8.8      8.600 7.4 352   8.500                                                                              7.2 347                           4.7   12      4.4      4.300 6.9 347  12.400                                                                              7.7 350                           5.0   25      0.9      0.825 6.1 344  15.600                                                                              8.0 353                           27.5  35      0.2      0.033 5.2 342  16.400                                                                              8.3 355                           ______________________________________                                    

Thus the desalted plasma in the concentrating stream has been broughtback to a conductivity value comparable to the original unsaltedcitrated plasma; and the water balance has been restored.

EXAMPLE II

Although the ED stack employed in example I contained ion-selectivemembranes (anion and cation types), the combination of ion-selectivemembranes with neutral (non-selective) membranes may also be used. Inthis example the stack of example I had its anion selective membranereplaced by a neutral membrane comprised of regenerated cellulose. Othertype neutral membranes well known in the art, such as reverse osmosis ordialysis type membranes could also be used if so desired. Neutralmembranes have the disadvantage of not being as efficient as asion-selective membranes. However, in processes where the energy input isnot a significant consideration, such membranes can then be utilized toadvantage.

In this example the stack containing the regenerated cellulose membranewas operated using substantially the same solutions and conditions asnoted in example I. The following table summarizes the course of thisrun:

    ______________________________________                                                                Conc. Stream                                                      Diluting Stream                                                   ______________________________________                                         (desalted plasma)                                                                        (fresh plasma)                                                                            Con-                                                                        Conduc-         duc-                                          Time            tivity          tivity                                  CD/K  (min.)  Amps    (K)    pH  vol. (K)   pH  vol.                          ______________________________________                                        3.3    0      12.0    16.500 8.2 360   0.050                                                                              5.3 340                           3.3   14      6.2      8.700 7.4 350   8.480                                                                              7.4 350                           3.3   27      3.1      4.300 6.9 342  12.390                                                                              7.7 355                           3.3   57      0.6      0.830 6.1 337  15.450                                                                              8.0 360                           23.3  80      0.2      0.039 5.2 334  16.420                                                                              8.2 363                           ______________________________________                                    

Here again, the desalted plasma has been restored to a conductivitycomparable to the original fresh plasma and has also had the waterbalance restored. It will be noted that the increased time of operation(80 minutes) was due to the current efficiency being considerably lessfor the combination of neutral and cation exchange membranes.

EXAMPLE III

This example is similar to the example I above except that the desaltedplasma is used in the original diluting stream of example I and a watersolution containing the removed salts from a prior desalting run is usedin the original concentrating stream. The polarity of the current isreversed (thus converting the original concentrating chambers todiluting chambers and the original diluting chambers to concentratingchambers) and the salts from the salt water stream are transferred tothe desalted plasma (now the concentrate stream) to bring the salts ofthe plasma back to its original concentration.

EXAMPLE IV

This example illustrates the removal of immune globulins (Ig) as afunction of the degree of desalting.

The apparatus of example I was used with 200 ml of heparinized humanplasma employed in the diluting chamber. The temperature was in therange of 10°-26° C. and the CD/K value used was approximately 4 (ma/cm²/mS/cm). A fluid velocity of 25 cm/sec. was employed. The followingtable summarizes the results and shows that about 50% of the total Ig'sare removed while albumin removal is substantially unaltered after 99.7%desalting.

    ______________________________________                                                                     Proteins                                                                      remaining in supernatant                         Time         Conduc-  %      (mg/100 ml)                                      (min.)                                                                              pH     tivity   Desalting                                                                            IgG  IgA  IgM  Albumin                           ______________________________________                                        0     7.55   15.280   0      820  115  72   4,410                             0     7.55   13.720   0      750  85   50   3,900                             5     7.35   7.020    48.80  780  80   46   3,900                             8     7.10   3.710    72.96  700  75   36   3,900                             10    6.55   1.310    90.60  630  75   24   4,100                             13    5.40   0.447    96.70  570  55   12   4,000                             15    5.20   0.190    98.60  450  50   10   4,000                             17    4.90   0.047    *99.70 410  40   16   3,900                             19    5.00   0.028    99.80  410  45   12   3,800                             20    5.10   0.022    99.84  410  45   10   3,800                             ______________________________________                                         *Summary of % Ig's Removed After 99.7% Desalting                         

                                % removed (corrected                                                % removed for water transfer)                               ______________________________________                                        IgG       =       45.3      46.7                                              IgA       =       52.9      55.9                                              IgM       =       68.0      71.4                                              Total Ig's                                                                              =       47.3      49.7                                              ______________________________________                                    

EXAMPLE V

This example illustrates a further embodiment of the invention, wherealteration of pH to substantially the isoelectric point of a proteinwhich it is desired to remove can assist its precipitation. Continuationof the desalting of the plasma resulting from example IV will bring thepH down to the isoelectric point (pI) of albumin (about 4.9) thuscausing its precipitation. The albumin precipitate is separated byfiltration and then resuspended by the addition of salt. This additionis accomplished using ED by making the albumin rich material as the saltconcentrating stream thus resulting in a 3-5% isotonic albumin solution.This albumin is essentially free of immunoglobulins and their complexesand can be used as a plasma expander. Thus this is a preferred methodfor those cases where more than 40-50% removal of immune globulins isdesirable for "immunepheresis" (removal of immunoglobulins) forautoimmune diseases.

In another embodiment of the invention, fractionation is achieved by"salting-out" i.e. the use of salts such as (NH₄)₂ SO₄, Na₂ SO₄ etc.brought into the protein mixture by electrodialysis. The variousproteins will precipitate out at different salt concentrations andthereby lend themselves to fractionation. A distinct advantage ofaccomplishing this by ED instead of by direct addition of salts is thatED allows more controlled addition of salts, thus avoiding localconcentration gradients. Electrodialysis "salting-out" is also muchfaster when compared to dialysis alone where only diffusion (and not anelectric potential) is the driving mechanism. Comparable fractionationis achieved by ED at a much lower salt content compared to eitheraddition or dialysis addition of salt.

The process and apparatus employed for salting-out will be furtherdescribed hereinafter for the fractionation of blood plasma protein butis not to be understood as limited to plasma only. Two of the"salting-out" embodiments are shown in FIGS. 3 and 4. As shown in FIG.3, fresh plasma (15) is pumped as the concentrating stream (saltreceiving stream) through two electrodialysis stacks (4) and (11). Theresulting salted-out plasma (22) is passed into separation means (7)such as a centrifuge where removal of any precipitated protein (17) isaccomplished. The resulting supernatant (8) which becomes the dilutingstream to the ED stack (11) is essentially an albumin filtratecontaining the salting out agent (such as Na₂ SO₄) from which aprecipitated protein fraction (17) has been removed (e.g.immunoglobulins and fibrinogen). This stream is made to give up its salt(note the direction of vertical arrows) when a current is impressedacross the ED stack, that is, transfer the salting-out agent to a freshplasma stream (15) thus causing the precipitation of certain proteins(globulins) from said fresh plasma stream. In turn this dilute stream(8) becomes depleted of salt and becomes essentially an albuminsolution. The final or polishing ED stack (4) is shown separately but itcan be part of a single ED stack. This final polishing step may not berequired where the product (9) is allowed to have salts which are notobjectionable for infusion. A make up electrolyte (20) may be needed ifthe polishing ED step is required. Thus such a process is compatiblewith in situ operation for therapeutic plasma exchange and can becarried out not only in batches but also continuously.

Another variation of the salting-out embodiment of this invention isshown in FIG. 4. Here the salting-out agent (salt solution) is addeddirectly to the plasma (15) causing immunoglobulins to precipitatewhereby they are separated (7) and removed (17) as by filtration. Theresulting plasma (16) employed as the dilute stream is desalted by EDstack (4); with the concentrating stream (18) becoming essentially asolution of the salting-out agent. This concentrated solution (19) canbe added directly to fresh plasma (15) in a closed loop fashion.Electrolytes (20) and formed elements (3) may be added to the desaltedalbumin solution (9) before administering the solution (12) back to thedonor or patient (14). Although Na₂ SO₄ is preferred as the salting-outagent it must be understood that this invention is not limited to it.Other salts and their mixtures may be used such as K₂ SO₄, (NH₄)₂ SO₄,sodium citrate, phosphates, NaCl, KCl, acetates, etc. and theirmixtures. The amount of salt added will of-course depend upon thefractionation desired. The following example illustrates the separationof IgG from albumin.

EXAMPLE VI

300 ml of plasma was warmed to 28°-37° C. and a saturated solution ofNa₂ SO₄ (approximately 6 N) at 28°-37° C. was added at a rate of 10-15ml/min. while constantly and rapidly stirring the plasma mixture. Theamount of albumin and globulins (IgG) remaining in the supernatant wasdetermined during the salt addition as a function of salt (electrolyte)concentration in the supernatant.

FIG. 5 compares the results using various methods of adding Na₂ SO₄electrolyte and shows the approximate protein fractionation (albumin andIgG's) occurring at different electrolyte strengths. Also specificallyshown is the fractionation curve resulting when a salt mixture (6 N NaCland 6 N Na₂ SO₄) was employed. A comparison of salting-out byelectrodialysis, dialysis and direct salt addition is also illustrated.

The results show that to obtain about 80% removal of globulins (IgG's)from fresh plasma requires a 1.8 N. salt (Na₂ SO₄) concentration in theplasma (supernatant) in the case where direct salt addition is employed.Under these conditions however, there was also a simultaneous removal (aloss) of about 15% albumin. Where the direct addition employed a saltmixture (Na₂ SO₄ +NaCl) an 80% removal of globulins occurred at about a2.05 salt normality accompanied by only a 5% removal of albumin. Wherethe addition of salt (Na₂ SO₄) is accomplished by use of dialysis an 80%globulin removal was noted at about a 2.3 salt normality but at a lossof about 10% albumin. In comparing the use of salt addition byelectrodialysis (ED) it is noted that there is a 95% removal ofglobulins at a much lower salt normality (1.2 N) with less than 15% lossof albumin. In summary it appears that more complete removal ofglobulins accompanied with smaller losses of albumin can be accomplishedat the lower salt normalities when the salt addition is performed byelectrodialysis. An alternate procedure where so desired is to employ acombination of direct addition or dialysis of the salting out agentthereafter followed by ED treatment to remove the added salts therefrom.

EXAMPLE VII

This example illustrates the separation of antihemophilic factor (AHF)and fibrinogen from the plasma. Since the activity of AHF is time andtemperature sensitive, the separation is carried out at a lowtemperature (4° C.). One procedure applied in the separation is loweringthe ionic strength of plasma preferably by ED desalting to cause theprecipitation (separation) of fibrinogen and AHF, later resolubilizingthe precipitate in for example, 0.15 N NaCl and thereafter subjectingthe resulting resolubilized liquid once again to ED to lower the ionicstrength thus causing the precipitation (and separation) of AHF fromfibrinogen. Alternatively after resolubilizing, the latter separationcan be caused by specific adsorption of AHF on anion exchange column orby gel permeation techniques.

A second procedure consists of the direct salting out of AHF at anappropriate salt strength. These procedures are also applicable for online as well as off line use as in the case of immunepheresis.

Another embodiment of this invention is the application of ED to thefractionation of plasma proteins using small amounts of low toxicityheavy metal ions such as zinc diglycinate as the precipitating agent.The plasma is first partially electrodialyzed to remove clotting factorswhich become precipitated during the desalting. These precipitatedfactors are removed and the resulting supernatant is passed through thesalt concentration compartments of an ED stack containing zincdiglycinate in the diluting compartments. On application of a electricalpotential a controlled amount of zinc diglycinate is transferred intothe concentrating compartments to give an ionic strength of about 0.10normal in the supernatant. The operation is carried out at about 0°-4°C. and at a pH of about 7.0-7.2. This results in the formation of aprecipitate consisting essentially of globulins with a supernatant richin albumin. The supernatant can be clarified of the added zinc bydesalting by ED after bringing the pH down to about 5.1-5.8 by suitableaddition of a buffer.

Aluminum chloride may also be substituted for zinc diglycinate forfractionally precipitating all proteins except γ-globulins. The directaddition of an equal volume of 0.1 M AlCl₃ at 0° C. to plasma with rapidstirring will precipitate all other proteins which may then beredissolved in 0.15 N. NaCl.

EXAMPLE VIII

This example illustrates the use of alternating current rather than theuse of direct current which is normally employed in electrodialysisoperations. The stack employed a combination of neutral (N) andcation-selective (C) membranes in an arrangement illustrated in FIG. 6.An alternating current ED stack is fully described in U.S. Pat. No.2,955,999 (C. E. Tirrell).

A valve metal such as niobium (4) plated with a noble metal such asplatinum (5) is used as an anode (1) and the valve metal (6) without thecoating of a noble metal is used as a cathode (3). The valve metals havethe property of conducting current only when they are made cathodic,hence effecting a sort of "rectification" of the alternating current(AC). Such a stack uses two cell pairs (instead of one as used inexample II) separated by a middle bipolar electrode (2) platinized (5)one one side to serve as an anode.

During the positive (+) current half cycle the platinized side (5) ofelectrode (1) serves as the anode and the unplatinized side (4) ofelectrode (2) as cathode and hence the membrane stack bound byelectrodes (1) and (2) is in service whereas the membrane stack betweenelectrodes (2) and (3) is inactive since electrode (3) beingunplatinized cannot function as an anode.

During the negative (-) current half cycle the stack between electrodes(2) and (3) is functional since the platinized side (5) of electrode (2)is anode.

Such a stack operates in a similar fashion as that of example II. Theapparatus will have the advantage of being operated from alternatingcurrent obviating the necessity of a rectifier. The neutral membrane canalso be replaced by an anion membrane to make the operation more energyefficient.

EXAMPLE IX

This example illustrates the removal of Factor VIII from human plasma byemploying electrodialysis for the desalting. The membrane separationapparatus used is a Dial-A-Cell™ stack commercially available fromIonics, Inc. of Watertown, Mass. and is fully described in U.S. Pat. No.4,202,772. The stack comprised two cell pairs having an effectivemembrane area of 13.6 cm². The ion-exchange membranes used were thecation selective (CR 61 CZL) and anion selective types (AR 103 QZL) bothalso obtainable from Ionics, Inc.

30 ml of fresh plasma containing ACD (anticoagulant solution consistingof a mixture of sodium citrate, citric acid and dextrose) was used inthis run. The starting plasma had a Factor VIII activity of 78% of thenormal. The following table summarizes the results of the desalting run.As the plasma is desalted, Factor VIII is precipitated out and hence thesupernatant is depleted in Factor VIII. At about the 90% desalting levelthe supernatant retains about 5-10% of Factor VIII, hence the removedprecipitate would contain about 90-95% of the Factor VIII originallycontained in the starting plasma sample.

    ______________________________________                                        Time  Conductivity          Factor VIII activity in                           (min.)                                                                              (K)        % Desalting                                                                              supernatant (% or normal)                         ______________________________________                                         0    13.6       0          78                                                12    11.6       14.7       70                                                18    8.4        38.2       63                                                24    6.4        52.9       42                                                30    3.6        47.6       30                                                33    2.8        79.4        9                                                36    2.2        83.8       12                                                39    1.8        86.8        5                                                42    1.0        92.6       11                                                ______________________________________                                    

The treatment of liquid whey to increase the desirable protein contentand decrease the ash (salt) and lactose components has been the objectof a variety of processes. L. H. Francis in U.S. Pat. No. 3,615,664discloses a technique in which lactose is removed from whey byconcentration of the raw whey to crystallize lactose and then subjectingthe supernatant to electrodialysis to effect demineralization. The sameinventor in U.S. Pat. No. 3,447,930 describes another process wheredemineralizing is done first followed by delactosing. These and otherprior art methods are directed to the purpose of obtaining a refinedhigh protein whey end product. Some of the major concerns in carryingout these processes are denaturation of whey protein (lactalbumin)during the application of heat to effect concentration andcrystallization. The process of the present invention is to overcomethese problems by salting-out the whey proteins, followed by theseparation and removal of the precipitated proteins by centrifugation orfiltration and thereafter removal of the salt from the resultingsupernatant. The supernatant will be comprised mainly of lactose andhence can be subjected to high temperatures without fear of proteindenaturation. It should be noted that this process not only separatesthe proteins but also effects desalting by methods previously describedin examples directed to plasma protein treatment.

EXAMPLE X

The ED apparatus used is similar to the one described in example I. Thediluting stream is comprised of 500 ml. of supernatant obtained from aprior whey run where substantially all whey proteins were removed i.e.salted-out by electrodialysis at about a 3.5 sodium sulfate normality atan operating temperature of about 38° C. 300 ml of concentrated wheywith a solid content of 22.5% (solids=12% proteins, 80% lactose hydrateand about 8.0% ash) is used as the concentrating stream. A directcurrent density of about 130 ASF is used (starting CD/K=2.0) and nearthe end of the run where the diluting stream becomes depleted of much ofits salts, the current is adjusted to conform to a CD/K of about 4.8.

The run is continued until a normality of about 3.5 Na₂ SO₄ is obtainedin the concentrating stream where the conductivity is about 95 milliSiemens/cm. The diluting stream volume which is reduced to about 300 mlis 90% salt free and may be further treated to recover lactose. Theconcentrating stream which increases in volume to about 500 ml developsa fine precipitate (turbidity) of protein which is removed bycentrifugation. The resulting highly salted supernatant is then used asthe diluting stream to transfer its salting-out agent to a next freshbatch of concentrated whey. This method of transfering the salting-outagent is accomplished in a manner similar to the cases of human plasmaprotein separation as described previously.

While the invention has been herein shown and described in what ispresently conceived to be the most practical and preferred embodimentsthereof, it will be apparent to those of ordinary skill in the art thatmany modifications may be made thereof within the scope of theinvention, which scope is to be accorded the broadest interpretation ofthe appended claims so as to encompass all equivalent assemblies andmethods.

The embodiments of this invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A process for separatingan aqueous protein mixture into fractions having intrinsicallydistinguishable compositions comprising removing substantially all theturbidity therefrom, subsequently decreasing the salt content thereof bypassing said mixture at a velocity of between 3-40 cm/sec. into and outof an electrodialysis apparatus containing at least one pair ofcontiguous membranes defining a liquid flow chamber therebetween,impressing an electric current across said apparatus at about a CD/K=0.1to 10 (where CD is current density in m-amps/cm² and K is theconductivity of the aqueous mixture in milli Siemens/cm) therebyaltering the ionic environment of said mixture by decreasing its ionicconcentration sufficiently to at least partially destabilize one or moreproteins in said mixture, allowing said destabilized protein to formturbidity, subsequently removing substantially all of said turbidity andmaintaining the temperature of said mixture during the said separationin the range of between about 0°-40° C.
 2. A process according to claim1 wherein the ionic environment of said mixture is altered by decreasingits ionic concentration by at least about 10 percent.
 3. A processaccording to claim 1 wherein the ionic environment of said mixture isaltered by changing its pH substantially toward the isoelectric point ofat least one of the said proteins.
 4. A process according to claim 1wherein the membranes of said electrodialysis apparatus aresubstantially non-ion selective.
 5. A process according to claim 1wherein at least one of the said membranes in every contiguous pair ision selective.
 6. A process according to claim 5 wherein at least asubstantial fraction of the electric current is direct current.
 7. Aprocess according to claim 1 wherein one of the membranes in everycontiguous pair is cation selective.
 8. A process according to claim 1wherein one of the membranes in every contiguous pair is cationselective and the other is anion selective.
 9. A process according toclaim 1 wherein said subsequently removed turbidity is resolubilized.10. A process according to claim 1 wherein said subsequently removedturbidity is at least in part resolubilized and the said resolubilizedpart is converted to a turbid insoluble fraction and a soluble fractionby again decreasing the ionic concentration by electrodialysis and theresulting turbid insoluble and soluble fraction are separated from eachother.
 11. A process according to claim 1 wherein said subsequentlyremoved turbidity is at least partly resolubilized in electrolyte atleast in part recovered by electrodialysis from said protein mixture andsaid resolubilized protein is then separated into at least twointrinsically distinguishable components.
 12. A process according toclaim 1 wherein said aqueous protein mixture comprises plasma proteinsand the said subsequently removed turbidity comprises fibrinogen andantihemophilic factor.
 13. A process according to claim 1 wherein saidaqueous protein mixture comprises substantially undenaturated plasma andthe said subsequently removed turbidity comprises immunoglobulins.
 14. Aprocess according to claim 1 wherein said aqueous protein mixturecomprises plasma proteins, the subsequently removed turbidity comprisesfibrinogen and antihemophilic factor which turbidity is resolubilizedand separated into a fibrinogen rich fraction and an antihemophilicfactor rich fraction by contacting with a material selected from thegroup consisting of ion exchange resin granules, gel permeation granulesand mixtures of the same.
 15. A process according to claim 1 wherein theaqueous protein mixture comprises plasma proteins and the subsequentlyremoved turbidity comprises albumin.
 16. A process according to claim 15wherein the subsequently removed turbidity is resolubilized for use as aplasma expander.
 17. The process according to claim 1 wherein saidaqueous protein mixture comprises whey.
 18. A process according to claim1 wherein said aqueous protein mixture comprises plasma proteins and thesaid subsequently removed turbidity comprises antihemophilic factor.